Safety pharmacological studies are non-clinical studies to examine the safety of novel drugs on human from pharmacological viewpoints. Guideline on Safety Pharmacology Studies which aims at examining the safety of test substances on human and predicting adverse effects thereof has been set in the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) between Europe, Japan and the United States. According to the Guideline, examination of the arrhythmogenic effect, in particular, the presence or absence of electrocardiographic QT interval prolongation effect, of test substances is required as a part of safety pharmacological studies. In order to protect patients from ventricular tachycardia, torsades de pointes and lethal arrhythmia associated with QT interval prolongation induced by drug administration, it is very important in the development of drugs to detect QT interval prolongation effect which may induce such serious adverse effects.
To date, it has been known that a large number of drugs having QT interval prolongation effect inhibit delayed rectifier potassium channels in cardiomyocytes. It is believed that hERG (human ether-a-go-go related gene) channel is functioning as a major constituent protein in the delayed rectifier potassium channel. Therefore, in the draft guideline for non-clinical evaluation of the potential for delayed ventricular repolarization (QT Interval Prolongation) by human pharmaceuticals (ICH-S7B), ion channel assay using hERG channel-transferred cells is recommended as a non-clinical study.
Conventionally, as a method of appropriately evaluating the effect of a compound on hERG channels, the patch clamp technique has been used in which channel activities of cells that had been allowed to express hERG channels or cardiomyocytes inherently possessing hERG channels are recorded by direct use of glass microelectrodes to the cells or cardiomyocytes (Neher, E. and Sakmann, B., Nature, Vol 260, 779-802, (1976)). However, though this patch clamp technique enables appropriate evaluation of drugs on hERG channels with high accuracy, it has the following problems. That is, it requires highly skilled technology, has a remarkably low throughput (1-5 compounds/day/person) and thus is remarkably insufficient in throughput for a great number of candidate compounds at the development stage of drugs.
On the other hand, as methods for enhancing the throughput of hERG channel evaluation, a method of detecting the release of a radioactive isotope Rb3+ (Cheng, C. S. et al., Drug Dev. Ind. Pharm. vol. 28, 177-191, (2002)), a method in which competitive binding to a radioactive isotope tritium (3H)-labeled dofetilide is observed and evaluated (Finlayson, K. et al. Eur. J. Pharm. vol. 430, 147-148 (2001)) and a method using membrane potential sensitive dyes (Tang, W et al. J. Biomol. Screen vol. 6, 325-331 (2001); Baxter, D. F. et al. ibid. vol. 7, 79-85 (2003)) have been reported. However, these methods are complicated in operations because radioactive substances are used. Besides, since they are indirect methods, their sensitivity and accuracy are low and far from the measurement accuracy that can be obtained by the patch clamp technique.
Recently, fully automated high throughput patch clamp systems which evaluate the influence by hERG channels appropriately and at the same time have a high throughput have been developed as means to solve the above-described problems. Some of such instruments have been commercialized (IonWorks HT™ from Molecular Device; PatchXpress™ 7000A from Axon Instruments). These apparatuses are intended to suspend hERG channel-expressing cells, suck the cells into a small hole located at the center of each well utilizing dropping by gravity and negative pressure, and to thereby make the cells capable of current recording. Therefore, the highly skilled technology required in the patch clamp technique is not necessary. Besides, the throughput of compounds is very high; data from more than 3000 points can be obtained per day. However, for making evaluation with such an apparatus, hERG channel-expressing cells used in experiments are extremely important. While it is possible to select hERG channel high expressing cells for measuring in the conventional patch clamp technique where a single cell is selected and evaluated, it is impossible to select high expressing cells in measurement with a fully automated high throughput patch clamp system because cells present are used at random. Therefore, in order to evaluate multiple samples accurately with a fully automated high throughput patch clamp system, more than a specific ratio of cells must be expressing hERG channels and yet the expression levels must be sufficient. Various attempts have been made to prepare hERG channel-expressing cells which can be used in this fully automated high throughput patch clamp system.
According to reports so far made, stably expressing cells were obtained by introducing hERG gene into cells by transfection using the calcium phosphate method or lipofection and then performing extremely labor-consuming procedures such as cloning cells by the limiting dilution culture method and confirming the quantity of transferred hERG gene, or measurement of currents through hERG channels (Tang, W. et al. J. Biomol. Screen, vol. 6 325-331 (2001); Assay and Drug Development Technologies, 1(2-3), 127-135, 2003). However, these techniques cannot secure a sufficient quantity of transferred hERG gene even by spending a great labor of cloning by the limiting dilution culture method, which results in a very weak hERG current per cell. When the hERG current is small, it is impossible to obtain a sufficient S/N ratio and, as a result, measurement sensitivity decreases. Further, even when a cell with whatever large hERG current has been obtained by cloning, it is often difficult to measure the amplitude of the hERG current with high sensitivity with a fully automated high throughput patch clamp system.
In measurement of currents in the ion channel into which hERG channel is classified, influence of the ion channel which cells endogenously have varies by cell species. Therefore, the practical value of measurement often varies greatly by changing cell species. For this reason, it was also necessary to repeat labor-consuming operations such as described above when measurement was performed with a different cell species.